The invention relates to a novel class II PI3 kinase, termed PI3K-C2xcex1, and in particular the isolation thereof having regard to its relevant sequence structure and/or biochemical characteristics; means used in the isolation or production thereof; antibodies adapted to bind thereto; and assay kits relating thereto.
Phosphoinositides have been implicated in a variety of cellular processes as diverse as vacuolar protein sorting (1,2), cytoskeletal remodelling (3) and mediating intracellular signalling events through which growth factors, hormones and neurotransmitters exert their physiological effects on cellular activity, proliferation and differentiation (4,5,6).
Recently a family of proteins have been cloned and characterised and shown to be enzymes catalysing the addition of phosphate to inositol. Eukaryotic cells contain a variety of inositol derivatives phosphorylated to different extents. PtdIns(3)P is constitutively present in eukaryotic cells and its levels are constant upon extracellular stimulation. PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (7,8) are virtually absent in resting cells but are rapidly induced upon stimulation with a variety of ligands. The enzymes catalysing these reactions are phosphoinositide lipid kinases (hereinafter called PI3 kinases). A brief overview of the current data in relation to PI3 kinases classifies these enzymes into three distinct groups being designated to an individual class by their in vitro substrate specificity, biochemical characteristics and, in examples where a definitive function has been assigned, the nature of the biochemical activity regulated by the specific kinase.
PI3 kinase class 1 polypeptides have a broad spectrum activity, phosphorylating inositol lipids PtdIns, PtdIns(4)P and PtdIns(4, 5)P2. Class I kinases are subdivided into Class IA and IB. Class IA polypeptides include p110xcex1 (9), p110xcex2 (10) and p110xcex4 (11) which interact physically with the adaptor sub-unit protein p85. Moreover, p110xcex1 and p100 have a broad distribution in terms of expression pattern. p110xcex4 expression seems to be restricted to white blood cells. Class 1B includes p110xcex3 (12) which functions independently of p85. Although each of these Class 1 kinases catalyse phosphate addition to inositol lipid, the mechanism via which these enzymes are activated and regulated is achieved by different molecular mechanisms.
Class II PI3 kinases have a restricted substrate specificity phosphorylating PtdIns and PtdIns(4)P but not PtdIns(4,5)P2. Each of the kinases of this class are characterised by a conserved C2 domain in the carboxyl terminal region of the protein. The presence of conserved motifs within the C2 domain indicates that this region may confer regulation via calcium and/or phospholipid. A comparison of the murine and Drosophila class II kinases mp170 and PI3Kxe2x80x9468D respectively reveals a high degree of homology in the kinase domain of these proteins. Significant divergence occurs at the amino terminal regions of these polypeptides suggesting that adaptor proteins interacting with these variable domains may regulate kinase activity. Class II PI3 kinases do not interact with p85.
The third class of PI3 kinase, class III PI3 Kinase, is related to the S.cerevisiae gene Vps34 (1). This kinase was originally isolated as a gene involved in regulating vesicle mediated membrane-trafficking in yeast. The human homologue of Vps34 is complexed with a ser/thr kinase called Vps15p (14,15). Of the three classes of PI3 kinase this has the most restricted substrate specificity being strictly limited to PtdIns.
A novel human class II PI3 kinase is herein described and termed human PI3K-C2xcex1. It is characterised as a class II kinase due to the presence of a conserved C2 domain found in murine and Drosophila class II PI3 kinases (FIG. 2), its apparent lack of a p85 binding site and a substrate specificity limited to PtdIns and PtdIns (4) P (FIG. 4). The polypeptide is unique in that this is the first human class II kinase to be described. It has significant divergence in the amino terminal region of the protein when compared to the mouse homologue of human PI3K-C2xcex1 (16). It is also, surprisingly, the first PI3 kinase to be isolated that has resistance to PI3 kinase inhibitors Wortmannin and LY294002 (FIG. 5).
The use of selected inhibitors has proved extremely useful in analysing intracellular signal transduction cascades. Inhibitors used at low concentrations probably result in the modification of a single protein""s function thereby allowing the dissection of signal transduction pathways. A good example of this is the use of Pertussis toxin which is a cell permeant agent (17). The agent undergoes endocytosis into intact cells and results in the ADP-ribosylation of specific GTP-binding or G-proteins. This modification uncouples these G-proteins from their receptors therefore interfering with the cell""s response to receptor stimuli. Wortnannin is another cell permeant inhibitor (18,19). It is a fungal metabolite and has been shown to have in vivo anti-inflammatory or immunosuppressive effects in animal models. Wortmannin was first shown to inhibit cellular responses to receptor stimulation in neutrophils. The drug inhibited the respiratory burst induced by ligands such as N-formyl-Met-Leu-Phe, (fMLP),C5a,leukotriene B4 or platelet-activating factors. Importantly, Wortmannin failed to inhibit cellular response to TPA suggesting differential responses to the drug. In particular, the fact that the stimulation of calcium mobilising receptors is resistant to Wortnannin suggests that intracellular signalling initiated by these receptors is controlled by a quite separate kinase cascade.
The identification of PI3 kinases as the target for Wortmannin came from in vitro metabolic labelling of guinea pig neutrophils with 32P to monitor the uptake of phosphate into phospholipids in the presence of specific kinase inhibitors (20). In control experiments stimulation of fMLP receptors resulted in 32P labelling of phosphatidic acid and PtdIns(3,4,5)P3. The presence of Pertussis toxin had no effect on the phosphorylation of these phospholipids. However, pre-incubation of leucocytes with Wortmannin resulted in inhibition of 32P incorporation into only PtdIns(3,4,5)P3. Since this is the product of PI3 kinase catalysed reactions it seems likely that Wortnannin was specifically targeting PI3 kinase. Subsequently, Wortmannin has been shown to block a number of physiological processes including many insulin stimulated actions that would result in enhanced glucose utilisation (21). Wortnannin has proved to be an effective inhibitor of mammalian PI3 kinases. To date, no human PI3 kinase has been cloned and shown to be resistant to this agent.
The isolation and sequencing of an as yet unidentified human PI3 kinase that has significant homology to previously identified murine and Drosophila class II kinases is described (16,22,23). Comparison of the optimal alignment of these aforementioned proteins shows the human protein to be 32.5% homologous with sequences of the Drosophila PI3Kxe2x80x9468D and cpk proteins and 90.8% and 90.2% with the murine proteins mp170 and mcpk, FIG. 2. The carboxyl terminal region of the aforementioned proteins have increased homology due to the presence of a conserved C2 domain. This domain is characterised by the presence of motifs likely to be involved in the modulation of kinase activity by calcium and/or phospholipid.
The amino terminal region of human PI3K-C2xcex1 is extended by 176 amino acid residues which are lacking in the murine sequence of mp170. The murine mcpk protein has a 28 amino acid residue deletion in this region that is absent from human PI3K-C2xcex1 and a mp170. This divergence in sequence may be explained by the presence of the unique binding sites for adaptor proteins that regulate kinase activity. This is supported by the lack of an apparent p85 binding motif.
Furthermore, immunofluoresence experiments using a monoclonal antibody to the amino terminal glu-tag of PI3K-C2xcex1 reveals a punctate cytoplasmic distribution in Cos cells expressing the recombinant glu-tagged PI3K-C2xcex1 protein, FIG. 9. Moreover subcellular fractionation of Monkey Kidney Cos cells shows that epitope tagged PI3K-C2xcex1 is tightly associated with phospholipid containing membranes. The nature of this interaction is currently unknown but may be mediated via phospholipid interaction with the C2 domain or alternatively through binding to an additional polypeptide localised to microsomes and plasma membranes.
The recombinantly produced PI3K-2Cxcex1 kinase shows increased resistance to the general PI3 kinase inhibitors Wortmannin and LY294002, (FIG. 5) To date, all cloned human PI3 kinases have been found to be sensitive to these inhibitors. Studies of the Wortrnannin sensitive class I PI3 kinase p110xcex1 identifies residue lys-802 as being the site of Wortmannin covalent inactivation of kinase activity, a residue near or at the active site and involved in phosphate transfer. This residue is present in all PI3 kinase family members and also in PI3K-2Cxcex1, therefore the resistance to Wortnannin by PI3K-2Cxcex1 is not apparent.
Additionally it will be apparent that alternate methods for use in modulating the activity of PI3K-2Cxcex1 exists.
For example, having regard to the sequence data provided herein it is possible to provide antisense material which prevents the expression of PI3K-2Cxcex1.
As mentioned above, the invention embraces antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding a PI3K-2Cxcex1 protein, to decrease transcription and/or translation of PI3K-2Cxcex1 genes. This is desirable in virtually any medical condition wherein a reduction in PI3K-2Cxcex1 gene product expression is desirable, including to reduce any aspect of a tumor cell phenotype attributable to PI3K-2Cxcex1 gene expression. Antisense molecules, in this manner, can be used to slow down or arrest such aspects of a tumor cell phenotype.
As used herein, the term xe2x80x9cantisense oligonucleotidexe2x80x9d or xe2x80x9cantisensexe2x80x9d describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and thereby, inhibits the transcription of that gene and/or the translation of that mRNA. The antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based upon the DNA sequence presented in FIG. 1 or upon allelic or homologous genomic and/or DNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention. In order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides should comprise at least 7 (Wagner et al., Nature Biotechnology 14:840-844, 1996) and, more preferably, at least 15 consecutive bases which are complementary to the target. Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases. Although oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5xe2x80x2 upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3xe2x80x2-untranslated regions may be targeted. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs. In addition, the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol. Neurobiol. 14(5):439-457, 1994) and at which proteins are not expected to bind. Finally, although FIG. 1 discloses cDNA sequence, one of ordinary skill in the art may easily derive the genomic DNA corresponding to the cDNA of FIG. 1. Thus, the present invention also provides for antisense oligonucleotides which are complementary to the genomic DNA corresponding to FIG. 1. Similarly, antisense to allelic or homologous DNAs and genomic DNAs are enabled without undue experimentation.
In one set of embodiments, the antisense oligonucleotides of the invention may be composed of xe2x80x9cnaturalxe2x80x9d deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5xe2x80x2 end of one native nucleotide and the 3xe2x80x2 end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage. These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors.
In preferred embodiments, however, the antisense oligonucleotides of the invention also may include xe2x80x9cmodifiedxe2x80x9d oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness. The term xe2x80x9cmodified oligonucleotidexe2x80x9d as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5xe2x80x2 end of one nucleotide and the 3xe2x80x2 end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, phosphate triesters, acetamidates, peptides, and carboxymethyl esters.
The term xe2x80x9cmodified oligonucleotidexe2x80x9d also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3xe2x80x2 position and other than a phosphate group at the 5xe2x80x2 position. Thus modified oligonucleotides may include a 2xe2x80x2-0-alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. Modified oligonucleotides also can include base analogs such as C-5 propyne modified bases (Wagner et al., Nature Biotechnology 14:840-844, 1996). The present invention, thus, contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding PI3K-C2xcex1 proteins, together with pharmaceutically acceptable carriers.
Antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art. The compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient. The term xe2x80x9cpharmaceutically acceptablexe2x80x9d means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term xe2x80x9cphysiologically acceptablexe2x80x9d refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
It follows from the foregoing description that it is an object of this invention to isolate the nucleic acid sequence and derived amino acid sequence of the first human class II PI3 kinase.
It is a further object of this invention to recombinantly manufacture human PI3K-C2xcex1 kinase.
It is a further object of this invention to use computer assisted three dimensional reconstruction of human PI3K-C2xcex1 kinase protein to facilitate the identification of agonists or antagonists of PI3K-C2xcex1.
It is a yet further object to use said three dimensional reconstruction to design ligands that may modulate the biochemical activity of human PI3-C2xcex1kinase.
It is yet a further object of this invention to manufacture an antibody, preferably monoclonal, to PI3K-C2xcex1, and more preferably further still to manufacture a humanised antibody to PI3K-C2xcex1.
It is yet a further object of this invention that the monoclonal antibody be specific to class II PI3 kinases.
It is yet a further object of this invention to provide an assay kit for use in identifying said protein.
According to the first aspect of the invention there is provided the isolated nucleic acid molecule and/or corresponding protein of human PI3K-C2xcex1 as shown in FIG. 1, including any deletions, additions or substitutions, or a complementary sequence thereto.
In a preferred embodiment of the invention said complementary sequence binds to said nucleic acid sequence under stringent conditions. Preferably, these conditions include 0.5M sodium phosphate, pH7.2, 7% SDS, 1 mM EDTA at 65xc2x0 C.
In yet a further preferred embodiment of the invention said deletions, additions or substitutions do not alter the function of the protein or indeed do not alter the function in a deleterious manner thus, in one example, a nucleic acid molecule may be provided that binds to the relevant gene to prevent expression of P13K-C2xcex1.
In yet a further preferred embodiment of the invention said deletions, additions or substitutions do alter the function of the protein. Thus, in one example, a mutation in P13K-C2xcex1 could produce a dominant negative mutation that results in the inactivation of endogenous P13K-C2xcex1 in transfected or transformed cells.
According to a second aspect of the invention there is provided an antibody, or at least an effective part thereof, which binds at least with a selective part of said human PI3K-C2xcex1.
Ideally said effective part comprises FAb fragments.
Ideally said antibody is polyclonal or monoclonal and ideally farther still said antibody is specific to class II PI3 Kinases. Ideally said antibody is humanised by recombinant methods to contain the variable region of said antibody with an invariant or constant region of a human antibody.
Ideally said antibody is provided with a marker including a conventional label or tag, for example a radioactive and/or fluorescent and/or epitope label or tag.
More ideally still said humanised monoclonal antibody to class II PI3-kinases is produced as a fusion polypeptide in an expression vector suitably adapted for transfection or transformation of prokaryotic or eukaryotic cells.
According to a yet further aspect of the invention there is provided a construct including all, or part of, the said nucleic acid molecule of the invention.
In a preferred embodiment of the invention said construct is adapted to be a cloning vehicle for human PI3K-C2xcex1.
In a preferred embodiment of the invention said cloning vehicle may be used for expression of the protein sequence of human PI3K-C2xcex1 in transformed or transfected prokaryotic and eukaryotic cells. Preferably said cloning vehicle is adapted for use in E.coli, mammalian, insect, amphibian or fungal cells.
In a preferred embodiment of the invention said construct includes nucleic acid encoding at least a part of human PI3K-C2xcex1 which is functionally linked to a promoter so as to provide for either constitutive or inducible expression of human PI3K-C2xcex1 kinase. Preferably further still said construct is adapted for cell/tissue specific expression of PI3K-C2xcex1.
According to a yet further aspect of the invention there is provided cells transformed or transfected with the cloning vehicle of the invention and so including the nucleic acid sequence molecule encoding human PI3K-C2xcex1 or a part thereof.
According to a yet further aspect of the invention there is provided recombinantly manufactured human PI3K-C2xcex1.
According to a yet further aspect of the invention there is provided human PI3K-C2xcex1 kinases or a fraction thereof, which is characterised by insensitivity to the PI3 kinase(s) inhibitor Wortmannin and/or LY294002.
In a further preferred embodiment of this aspect of the invention said protein is also, or alternatively, characterised by the absence of a p85 binding site. In a further preferred embodiment of this aspect of the invention said protein is also, or alternatively, characterised by the absence of a type II polyproline motif present in Drosophilia P13Kxe2x80x9468D and cpk.
In a yet a further preferred embodiment of this aspect of the invention said protein is also, or alternatively, characterised by divergent amino terminal region when compared to other class II PI3 kinases.
In a yet a further preferred embodiment of this aspect of the invention said protein is characterised by a molecular weight of 190 Kda.
In yet a further preferred embodiment of this aspect of the invention said protein is also, or alternatively, characterised by an in vitro substrate specificity that is restricted to PtdIns, PtdIns(4)P or at least PtdIns, PtdIns(4)P.
In any of the above embodiments of the invention said fraction ideally comprises the full length amino acid sequence or truncations thereof.
According to a further aspect of the invention there is provided the isolated genomic DNA and/or cDNA of human PI3K-C2xcex1 kinase according to any previous aspect of the invention.
According to a yet further aspect of the invention there is provided a process for the isolation of human PI3K-C2xcex1 kinase which process includes a PCR reaction.
In a preferred process of the invention said PCR reaction comprises use of at least two primers adapted to bind to at least one selected part of nucleic acid of human PI3K-C2xcex1; and further said process also comprises the provision of conditions for amplifying at least one selected part of said nucleic acid using said primers; and further, following amplification, said process comprises the purification of the amplified product(s); and, optionally, isolation of at least one amplified fragment.
In a preferred process of the invention said primers are designed to selectively bind to the nucleic acid of class II kinases and ideally class II human kinases.
In yet a further preferred embodiment of the invention said primers are as follows:
sense 5xe2x80x2GGNGA T/C GA T/C T/C T A/G CGNCA A/G GA3xe2x80x2 (SEQ ID NO:4)
antisense 5xe2x80x2A/G AA A/G TGICC A/G AA A/G TC AIGIT AT A/G TG A/G TG A/G AA3xe2x80x2 (SEQ ID NO:5)
In yet a further preferred embodiment of the invention said primers include any one or more deletions, additions or substitutions which do not deleteriously affect the functional effectiveness of the primers.
According to a yet further aspect of the invention there is provided the use of any one of more of the aforementioned primers to isolate class II PI3 kinases and ideally human PI3K-C2xcex1.
According to a yet further aspect of the invention there is provided the use of any one or more of the aforementioned primers to isolate human PI3 kinases and ideally human PI3K-C2xcex1.
According to a yet further aspect of the invention there is provided an agent which is adapted to interact with the amino terminal region, or any part thereof, of human PI3K-C2xcex1 kinase.
According to a yet further aspect of the invention there is provided an assay kit for identifying human PI3K-C2xcex1 either intracellularly or in a cellular extract.
In a preferred embodiment of the invention said assay kit is a diagnostic kit.
Preferably said assay kit, or said diagnostic kit makes use of any one or more of the following techniques: western blotting; immunoprecipitation; immunofluorescence on whole cells or tissue sections; or quantitative ELISA reactions.
According to a yet further aspect of the invention there is provided a method for identifying agonists or antagonists effective at enhancing or blocking the activity of the polypeptide of the invention which comprises screening candidate molecules for such activity using the polypeptide, or fragments thereof the invention.
Ideally, screening may involve artificial techniques such as computer-aided techniques or conventional laboratory techniques.
Alternatively, the method of the invention may involve competitive binding assays in order to identify agents that selectively and ideally irreversibly bind to the polypeptide of the invention.
According to a yet further aspect of the invention there is provided a pharmaceutical or veterinary composition comprising an agent effective at enhancing or blocking the activity of the polypeptide of the invention which has been formulated for pharmaceutical or veterinary use and which optionally also includes a dilutant, carrier or excipient and/or is in unit dosage form.
According to a yet further aspect of the invention there is provided antisense oligonucleotide or a modified antisense oligonucleotide as herein described adapted to hybridise to at least part of the nucleic acid molecule shown in FIG. 1.
Thus, in general terms the invention describes the nucleic acid sequence and derived amino acid sequence of a novel human class II PI3 kinase, human PI3K-C2xcex1, which has novel features in relation to sequence, biochemistry and responses to well characterised inhibitors of PI3 kinases.